Bottom Line:
Our results evidenced that the ORF nature, position, and orientation induce strong discrepancies between the previously reported promoters' strengths and the observed ones.We conclude that, in the context of metabolic reconstruction, the strength of usual promoters can be dramatically affected by many factors.Among them, transcriptional interference and ORF nature seem to be predominant.

ABSTRACTFine tuning of individual enzyme expression level is necessary to alleviate metabolic imbalances in synthetic heterologous pathways. A known approach consists of choosing a suitable combination of promoters, based on their characterized strengths in model conditions. We questioned whether each step of a multiple-gene synthetic pathway could be independently tunable at the transcription level. Three open reading frames, coding for enzymes involved in a synthetic pathway, were combinatorially associated to different promoters on an episomal plasmid in Saccharomyces cerevisiae. We quantified the mRNA levels of the three genes in each strain of our generated combinatorial metabolic library. Our results evidenced that the ORF nature, position, and orientation induce strong discrepancies between the previously reported promoters' strengths and the observed ones. We conclude that, in the context of metabolic reconstruction, the strength of usual promoters can be dramatically affected by many factors. Among them, transcriptional interference and ORF nature seem to be predominant.

Mentions:
While chromosome integration of genes is the preferred technique for final, industrial strains, first optimization steps are usually performed on plasmids (Ro et al., 2006; Steen et al., 2008; Ukibe et al., 2009; Kocharin et al., 2012). Our goal was to assess whether the assigned strengths of promoters, when described in model systems, can be transposable when multiple expression cassettes are inserted on a single episomal plasmid. We therefore used as model a small, heterologous pathway leading to the synthesis of zeaxanthin, and implemented it in S. cerevisiae. The heterologous production of carotenoid molecules has previously been described using S. cerevisiae (Verwaal et al., 2007; Ukibe et al., 2009; Sun et al., 2012), demonstrating that rerouting the endogenous terpene pathway to these heterologous metabolites is feasible (Figure 1). Three heterologous ORFs from different carotenoid-producing organisms were used: CRTI (phytoene desaturase gene), CRTYB (bifunctional phytoene synthase and lycopene cyclase gene), both from Xanthophyllomyces dendrorhous, and CRTZ (β-carotene hydroxylase gene) from Pantoea ananatis. A small metabolic combinatorial library was built using six constitutive promoters that were reported to be strong, medium, or weak using the fluorescent protein GFP as a reporter (Sun et al., 2012). mRNA levels of the three abovementioned genes were quantified in various culture conditions.

Mentions:
While chromosome integration of genes is the preferred technique for final, industrial strains, first optimization steps are usually performed on plasmids (Ro et al., 2006; Steen et al., 2008; Ukibe et al., 2009; Kocharin et al., 2012). Our goal was to assess whether the assigned strengths of promoters, when described in model systems, can be transposable when multiple expression cassettes are inserted on a single episomal plasmid. We therefore used as model a small, heterologous pathway leading to the synthesis of zeaxanthin, and implemented it in S. cerevisiae. The heterologous production of carotenoid molecules has previously been described using S. cerevisiae (Verwaal et al., 2007; Ukibe et al., 2009; Sun et al., 2012), demonstrating that rerouting the endogenous terpene pathway to these heterologous metabolites is feasible (Figure 1). Three heterologous ORFs from different carotenoid-producing organisms were used: CRTI (phytoene desaturase gene), CRTYB (bifunctional phytoene synthase and lycopene cyclase gene), both from Xanthophyllomyces dendrorhous, and CRTZ (β-carotene hydroxylase gene) from Pantoea ananatis. A small metabolic combinatorial library was built using six constitutive promoters that were reported to be strong, medium, or weak using the fluorescent protein GFP as a reporter (Sun et al., 2012). mRNA levels of the three abovementioned genes were quantified in various culture conditions.

Bottom Line:
Our results evidenced that the ORF nature, position, and orientation induce strong discrepancies between the previously reported promoters' strengths and the observed ones.We conclude that, in the context of metabolic reconstruction, the strength of usual promoters can be dramatically affected by many factors.Among them, transcriptional interference and ORF nature seem to be predominant.

ABSTRACTFine tuning of individual enzyme expression level is necessary to alleviate metabolic imbalances in synthetic heterologous pathways. A known approach consists of choosing a suitable combination of promoters, based on their characterized strengths in model conditions. We questioned whether each step of a multiple-gene synthetic pathway could be independently tunable at the transcription level. Three open reading frames, coding for enzymes involved in a synthetic pathway, were combinatorially associated to different promoters on an episomal plasmid in Saccharomyces cerevisiae. We quantified the mRNA levels of the three genes in each strain of our generated combinatorial metabolic library. Our results evidenced that the ORF nature, position, and orientation induce strong discrepancies between the previously reported promoters' strengths and the observed ones. We conclude that, in the context of metabolic reconstruction, the strength of usual promoters can be dramatically affected by many factors. Among them, transcriptional interference and ORF nature seem to be predominant.